The long-term goals of this research are to explore the role of c-di-GMP in regulating bacterial signaling and to develop small molecules that interfere with c-di-GMP signaling pathways. C-di-GMP is a bacterial second messenger that commonly regulates biofilm formation in numerous human pathogens, including, among others, Vibrio sp., Salmonella typhimurium, Yersinia pestis, and Pseudomonas aeruginosa. Cellular levels of c-di-GMP are controlled by the opposing activities of diguanylate cyclases that synthesize c-di-GMP and phosphodiesterases that hydrolyze it. C-di-GMP, diguanylate cyclases, and c-di-GMP phosphodiesterases are not found in humans, making c-di-GMP signaling systems an attractive target for anti-bacterial intervention. Here we propose to determine X-ray crystal structures of diguanylate cyclases in complex with different digualylate cyclase inhibitors. The inhibitors were identified using a high-throughput in vivo genetic screen, demonstrated to specifically inhibit the enzymatic activity of multiple digualylate cyclases from different bacterial species in vitro, and shown to repress biofilm formation under static or flow conditions. The X-ray crystal structures will reveal the mechanistic basis of inhibitor function, e.g., whether the inhibitors are binding to the diguanylate cyclase active site and acting as competitive inhibitors, and also identify the inhibitor functional groups mediating the compound-target interactions. This information will drive structure-activity relationship studies designed t generate tighter binding antagonists through iterative rounds of compound redesign, synthesis, and in vivo genetic and in vitro biochemical analysis.